This invention relates to a new facultatively anaerobic microorganism for degrading recalcitrant toxic waste materials, and particularly chlorinated aromatic compounds, into materials which are more environmentally acceptable.
The chemical industry annually generates enormous quantities of synthetic chemicals such as dielectric fluids, flame retardants, refrigerants, heat transfer fluids, lubricants, protective coatings, pesticides, including herbicides and insecticides, as well as many other chemicals and petroleum products used in agriculture, industry and health care. While these materials are invaluable and sustain a high standard of living for the population, they are foreign to the biosphere and can cause serious problems when released into the environment. Other sources of toxic chemicals include the waste materials generated during the manufacture of such useful chemicals.
Large amounts of the toxic chemicals generated annually by the chemical industry accumulate in animal and plant tissues and can cause serious health problems. Since these chemicals are not products of natural processes, and may possess structural features which are not commonly found in nature, they tend to persist in the environment and are resistant to degradation from naturally occuring organisms. Halogenated aromatic compounds are known to be particularly hazardous and also strongly resistant to biodegradation due to their cyclic nature and low concentration in the environment. Therefore, they tend to persist and accumulate to dangerous levels. Some of these materials are toxic, mutagenic and/or carcinogenic at very low concentrations. Polychlorinated biphenols (PCB's), chlorinated phenoxyacetates, and chlorinated benzoic acids (CBA's) are examples of chlorinated aromatic compounds considered to be hazardous wastes and priority pollutants. These chemicals are introduced directly into the biosphere as herbicides, pesticides, electrical transformer fluids or land treatment systems, or indirectly from unsuccessful landfills, chemical spills, or as wastes from chemical manufacturing processes, depending on their production method, shipment, use and disposal. Human exposure to and concern over these chemicals has increased in recent years due to increasing population density and industrial activity. Chlorinated benzoic acids or chlorobenzoates are of particular concern since they are intermediates in the microbial metabolism of more complex chlorinated aromatic compounds.
A variety of microorganisms have been isolated that have the capability of efficiently utilizing aromatic organic chemicals as sole carbon sources for growth (e.g. toluene, phenol, and naphthalene). See Clarke, P. H. and Ornston, L. N. (1975) "Metabolic Pathways and Regulations", 1, 191-196 in Clarke, P. H. and Richmond, M. H. (ed.), "Genetics and Biochemistry of Psuedomonas", John Wiley, London. However, the corresponding chlorinated aromatic compounds (chlorotoluenes, chlorophenols, chloronephthalenes) are biodegraded very slowly, if at all. See Alexander, M. (1973) "Non-Biodegradable and Other Recalcitrant Molecules", Biotechnology--Bioengineering, 15: 611-647.
A possible reason for this recalcitrance is the reduced reactivity of halogenated aromatic rings. The aromatic ring must be cleaved for the cycling of carbon in the metabolism of aromatic hydrocarbons. The presence of a halogen substituent on the aromatic ring adversely affects degradation. A halogen is an electronegative substituent which lowers the electron density of sites around the aromatic ring thereby reducing the chemical reactivity of the compound, rendering the ring less susceptible to microbial attack. These steric effects are influenced by the nature, position, and degree of substitution. As the number of halogen substituents increases, the arylhalide becomes less susceptible to microbial attack.
Notwithstanding, microorganisms have been isolated from the environment that are capable of growth on chlorinated aromatic compounds. For example, Chakrabarty, A. M., (1976) "Plasmids in Pseudomonas"; Ann. Rev. Genet. 10, 7-30, discloses bacteria which utilize haloaromatic compounds and the degradative pathways of intermediates involved. Several other publications deal with the microbiodegradation of halogenated hydrocarbons. For example, Bourquin, A. W. and Gibson, D. T. (1978) "Microbial Degradation of Halogenated Hydrocarbons; Water Chlorination Environmental Impact and Health Effects", 2, 253-264 disclose various microorganisms such as Aspergillus sp., Achromobacter sp., Arthrobacter sp. and Clostridium sp., as useful for dehalogenation of various substrates such as 2-chlorophenoxyacetate, 2,4-dichlorophenol, 3-chlorobenzoate, hexachlorocyclohexane, and 4-chlorobenzoate. Gibson, D. T., Koch, J. R., Schuld, C. L. and Kallio, R. E. (1968)--Biochemistry, 7 No. 11 3795-3802 in their paper on "Oxidative Degradation of Aromatic Hydrocarbons by Microorganisms including the Metabolism of Halogenated Aromatic Hydrocarbons," disclosed Pseudomonas putida as useful in the degradation of toluene and chlorinated compounds such as halobenzenes and p-chlorotoluene and state that the presence of halogen atoms greatly reduces the biodegradability of aromatic compounds. They also disclose that microorganisms have been isolated that have the capability to cometabolize a chlorinated aromatic chemical during growth on its nonchlorinated analog. For example, the conversion of chlorotoluene to chlorocatechol during growth of Pseudomonas putida on toluene has been demonstrated. This organism would not further metabolize the chlorocatechol, rather it is known that other microorganisms possess the ability to metabolize chlorocatechols. See Dorn, E. M., Hellwig and Reineke, W. and Knackmuss, H. J. (1974), "Isolation and Characterization of a 3-Chlorobenzoate Degrading Pseudomonas", Arch. Microbiology 99, 61-70 and also see Evans, W. C.; Smith, B. S. W.; Fernley, H. N.; and Davies, J. I, (1971), "Bacterial Metabolism of 2,4-Dichlorophenoxy Acetate", Biochem J., 122, 543-55. Chlorocatechol is known to be an intermediate in many of the metabolic pathways for utilization of chlorinated aromatic compounds. The chlorocatechol is further metabolized with the subsequent removal of chlorine. See Tiedje, J. M.; Duxbury, J. J.; Alexander, M. and Dawson, J. E. (1969), 2,4 D Co-metabolism: Pathway of Degradation of Chlorocatechols by Arthrobacter, J. Agr. Food Chem, 17, 1021-2026. Hartmann, J., Reineke, W., Knackmuss, H. J., (1979) Applied & Environmental Microbiology; 37, No. 3, 421-428 show a species of Pseudomonas identified as sp. WR 912 capable of degrading chlorobenzoic acids. Shubert, R., (1979) Fed. Ministry for Research and Technology, Goethe University, Frankfurt, W. Germany in his paper on "Toxicity of Organohalogen Compounds", discloses the minimal inhibitory concentrations preventing growth of various bacteria including a Pseudomonas cepacia in various chlorinated compounds including chlorotoluene.
Cometabolism is effective in the biodegradation of haloaromatic xenobiotic compounds because they do not have to serve as a sole source of carbon and energy for the microorganisms. It allows a microbial population to eliminate the toxicity of a hazardous compound while growing on another. In addition, through a series of cometabolic reactions among different microorganisms, total degradation of a compound could occur. PCB's, chlorinated phenoxyherbicides, and chlorinated benzoic acids are all known to be degraded slowly through cometabolism.
The cometabolic theory was utilized to develop a technique termed analogue enrichment as a means of inducing microbial degradation of environmental pollutants. See Horvath, R. S. and Alexander, M. "Cometabolism of m-Chlorobenzoate by an Arthrobacter", Applied Microbiology 20, 254 (1970). This technique takes into account the fact that microorganisms will attack a normally non-biodegradable substance in the presence of a substrate which is similar in structure to the target compound. The analogue induces the necessary enzyme system for the degradation of the recalcitrant compound. Analogue enrichment increases the decomposition rate of the target compound.
It has been suggested that because halogenated compounds are not generally found in nature, microorganisms have not evolved which possess efficient enzyme systems or genes which express themselves for the degradation of such chemicals; see Chatterjee, D. K., Kellogg, S. T., Furukawa, K., Kilbane, J. J., Chakrabarty, A. M., "Genetic Approaches to the Problems of Toxic Chemical Pollution", Third Cleveland Symposium on Macromolecules, 1981. Chakrabarty disclosed a technique for artificially inducing the biodegradability of 2,4,5 trichlorophenyl acetic acid (2,4,5 T) by gradually exposing bacteria to increased concentrations of the chemical over the course of about one year; see Chatterjee, D. K., Kellog, S. T., Eatkins, D. R. and Chakrabarty, A. M. in "Molecular Biology, Pathogenicity and Ecology of Bacterial Plasmids", Plenum Publishing Corp., N. Y., 1981, pp. 519-528.
U.S. Pat. Nos. 4,477,570 and 4,493,895, issued Oct. 16, 1984 and Jan. 15, 1985, respectively, the disclosures of which are incorporated by reference herein, disclose strains of Pseudomonas cepacia which are aerobic microorganisms and possess the capability of biodegrading halogenated organic compounds such as chlorobenzoates and chlorotoluates. These microorganisms were isolated from soil samples obtained from a landfill site which had been used for the disposal of chlorinated organic wastes during the period 1955-1975, and are identified as ATCC 31939, ATCC 31940, ATCC 31941, ATCC 31942, ATCC 31943, ATCC 31944, and ATCC 31945, all based on deposits made at the American Type Culture Collection. The plasmids contained in these microorganisms which code for the degradation of chlorinated aromatic compounds were isolated and designated as pRO 4.7, pRO 31 and pRO 54. Other plasmids which code for the degradation of chlorinated aromatic compounds are shown in the following Table 1:
TABLE 1 ______________________________________ Plasmid Degradative Pathway ______________________________________ pAC21 p-chlorobiphenyl pAC25 3-chlorobenzoate pAC27 3- and 4-chlorobenzoate pAC29 3-, 4-, and 3,5-dichlorobenzoate pJR2 2,4-dichlorophenoxyacetate pAC31 3,5-dichlorobenzoate pKF1 chlorinated biphenyls pJP1 2,4-dichlorophenoxyacetate ______________________________________
The plasmids listed in Table I are found in such diverse microorganisms as Pseudomonas putida, Pseudomonas aeruginosa, Klebsiella pneumonia, Serratia manscescens, gram negative Acinetobacter and gram positive Arthrobacter.
Klebsiella pneumonia and Serratia marescens are facultatively anaerobic enteric bacteria which were isolated from PCB-contaminated sediment of the Hudsom River. These microorganisms harbor the pAC21 plasmid and are capable of metabolizing p-chlorobiphenyl as their sole source of carbon and energy. See Kamp, P. V. and Chakrabarty, A. M., "Plasmids Specifying p-Chlorobiphenyl Degradation in Enteric Bacteria", in Plasmids of Medical, Environmental, and Commercial Importance, Biomedical Press, Elsevier, North-Holland (1979).
Enteric bacteria are not known for their ability to utilize hydrocarbons since they do not express genes for hydrocarbon degradation in the laboratory. However, the Hudson River isolates catabolize p-chlorobiphenyl as well as 4-chlorobenzoic acid, p-hydroxybenzoate and 2,4-D. The pAC21 plasmid is believed to be responsible for this activity. Strong selective pressures including high, localized concentrations of a toxic substance and the river bottom environment favored the appearance of such novel microorganisms.
The use of microorganisms for the treatment of wastewater is an economical alternative to physical treatment systems since biological treatment involves lower capital investment, lower energy requirements, a self-sustaining operation, and finally, the possibility for product recovery. In addition, biological systems offer the possibility of treatment in pre-existing municipal waste facilities, thus lowering the initial capital investment even further.
One particular biological treatment system of current interest is the sequencing batch reactor (SBR), which is a fill and draw activated sludge system operated in a batch treatment mode and utilizing a single tank for equalization, aeration and sedimentation. The use of a sequencing batch reactor with an inoculum of microorganisms capable of degrading chlorinated hydrocarbons is described in U.S. Pat. No. 4,511,657, issued Apr. 16, 1985, the disclosure of which is incorporated by reference herein.
Although several types of microorganisms which demonstrate the capacity to use chlorinated aromatic compounds as their sole source of carbon and energy are known, most of these are aerobic microorganisms which require oxygen for growth. Consequently, these bacteria would not be suitable for use in oxygen lean environments such as subsoil environments and underwater sediments which can contain toxic chemicals or wastes resulting from spills or direct applications to the soil. A facultatively anaerobic microorganism which could survive with or without oxygen by shifting to different metabolic processes in each case and which has the capability of degrading haloaromatic compounds would be extremely useful for soil detoxification.